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Spliceosomal cleavage generates the 3′ end of telomerase RNA

Abstract

Telomeres cap the ends of chromosomes and provide a means to complete replication. The DNA portion of telomeres is synthesized by the enzyme telomerase using part of an RNA subunit as a template for reverse transcription. How the mature 3′ end of telomerase RNA is generated has so far remained elusive. Here we show that in Schizosaccharomyces pombe telomerase RNA transcripts must be processed to generate functional telomerase. Characterization of the maturation pathway uncovered an unexpected role for the spliceosome, which normally catalyses splicing of pre-messenger RNA. The first spliceosomal cleavage reaction generates the mature 3′ end of telomerase RNA (TER1, the functional RNA encoded by the ter1+ gene), releasing the active form of the RNA without exon ligation. Blocking the first step or permitting completion of splicing generates inactive forms of TER1 and causes progressive telomere shortening. We establish that 3′ end processing of TER1 is critical for telomerase function and describe a previously unknown mechanism for RNA maturation that uses the ability of the spliceosome to mediate site-specific cleavage.

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Figure 1: An intron in TER1 is essential for RNA maturation.
Figure 2: Splicing and the Sm binding site.
Figure 3: A direct role for the U1 snRNA in TER1 maturation.
Figure 4: The first and second transesterification reactions have opposing effects on TER1 maturation.
Figure 5: Requirements for 3′ end processing by the spliceosome.

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Acknowledgements

The authors thank H. Yang and the other members of the Baumann laboratory for help and discussions, Y. Tzfati and L. Tomaska for sharing results before publication, and A. Berglund, M. Blanchette, R. Conaway and T. Cech for discussions and comments on the manuscript. We also thank the Molecular Biology Core Facility for site-directed mutagenesis and sequencing, M. Gogol and R. Voelker for computational analysis, and D. Baumann and R. Helston for proofreading of the manuscript. This work was funded by the Stowers Institute for Medical Research and a Pew Scholars in the Biomedical Sciences Award to P.B.

Author Contributions P.B. made the initial observations, oversaw the project and designed the experiments. J.T.B. and P.B. developed protocols for RNA isolation, northern blotting and primer extension analysis. J.A.B. contributed plasmids and strains and performed telomere length analysis and RT–PCR assays. J.T.B. conducted northern blotting and primer extension analysis. W.T. characterized the TER1-Sm1 mutant. All authors contributed to data analysis, and P.B. wrote the manuscript.

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Correspondence to Peter Baumann.

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Supplementary Information

This file contais Supplementary Figures 1-6 illustrating the 5′ splice site consensus in S. pombe, the effects of mutations in TER1 on splicing, the mapping of the branch point by primer extension and TER1 processing at various levels of expression. A detailed schematic of the heterologous intron and mutations therein is also provided. (PDF 1177 kb)

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Box, J., Bunch, J., Tang, W. et al. Spliceosomal cleavage generates the 3′ end of telomerase RNA. Nature 456, 910–914 (2008). https://doi.org/10.1038/nature07584

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